Genome annotation and comparative genome analyses

Chloroplast genome functional annotation includes encoding gene prediction and noncoding RNA annotation (rRNA and tRNA annotations). Gene annotation was performed using CPGAVAS2 [31], and the map of the circular A. inebrians chloroplast genome was drawn through the online tool Chloroplot [32].

The distribution of codon usage was detected by using codonw (version 1.4.4; https://sourceforge.net/projects/codonw/) with the relative synonymous codon usage (RSCU) ratio [33]. The codon of A. inebrians chloroplast was visually compared among species of 17 Poaceae with r language and tbtools [34].

The A. inebrians chloroplast genome was compared with the other five chloroplast genomes using the Shuffle–Lagan model of the mvista program [35]; Alopecurus japonicus served as the reference. irscope was used to visualize the boundaries between the IR and SC regions of A. inebrians, and the results were compared and analyzed with three other Poaceae species [36]. The four chloroplast genomes of Poaceae were initially compared using mafft [37] and then manually adjusted using bioedit [38]. Variable sites and nucleotide variations (Pi) in the entire chloroplast genome and LSC, IR and SSC regions of four species were calculated using dnasp [39].